This invention relates generally to the field of cell cycle control and more specifically to the role of ubiquitination in the regulation of cell cycle progression.
The irreversible nature of proteolysis makes it well-suited to serve as a regulatory switch for controlling unidirectional processes. This principle is evident in the control of the cell cycle, where initiation of DNA replication, chromosome segregation, and exit from mitosis are triggered by the destruction of key regulatory proteins (Schwob et al., Cell 79:233-244, 1994; Glotzer et al., Nature 349:132-138, 1991; Cohen-Fix, et al., Genes Dev. 10:3081-3093, 1996). Proteins are typically marked for proteolytic degradation by attachment of multiubiquitin chains.
In the early 1970s a novel protein was extracted from bovine thymus which was thought to have properties relating to the differentiation of T and B lymphocytes. This same protein was later found to be not only in the thymus but in all other eukaryotic cells. Due to its ubiquitous nature, the new protein was named ubiquitous immunopoietic polypeptide (Goldstein et al., Proc. Natl. Acad. Sci. U.S.A. 72: 11-15, 1975). Another protein of similar molecular weight was also discovered, and seemed to be involved in the ATP dependant degradation of denatured globulin in reticulocytes (Ciechanover et al., Biochem. Biophys. Res. Commun. 81, 1100-1105, 1978).
This protein was a small protein made up of only 76 amino acids, and is now known as ubiquitin. Ubiquitin has many diverse functions, and is one of the most highly conserved sequences of all proteins found in eukaryotic cells, with only minor variations of two or three amino acids found between organisms as evolutionarily dissimilar as mammals, oats, and yeasts (xc3x96zkaynak et al., EMBO J. 6(5):1429-1439, 1987).
Ubiquitin may have many roles in cell function including the mediation of various stress responses, repair of damaged DNA, regulation of differential gene expression, modification of histones and receptors, effects in neurodegenerative diseases, and control of the cell cycle. Other novel functions also suggested include the behavior of ubiquitin in a xe2x80x98chaperone-likexe2x80x99 role in the assembly of ribosomal proteins and as a response to heat shock. However, the most important role appears to be the role ubiquitin plays in selective protein degradation. The ability of ubiquitin to target proteins for degradation gives it a key role in the regulation of the cell cycle.
Many of the enzymes involved in ubiquitin dependant proteolysis have been identified, and the mechanism by which certain proteins are degraded has been determined. The presence of at least two different components required for ubiquitin dependant proteolysis have been confirmed, and that the mechanism of degradation is known to require the utilization of energy obtained from ATP (Ciechanover et al., Proc. Natl. Acad. Sci. U.S.A. 77:1365-1368, 1980).
The first step in selective degradation is the ligation of one or more ubiquitin molecules to a protein substrate (Hershko et al., Proc. Natl. Acad. Sci. U.S.A. 77:1783-1786, 1980). This process is initiated by ubiquitin-activating enzyme (E1), which activates ubiquitin by adenylation and becomes linked to it via a thiolester bond. Ubiquitin is then transferred to a ubiquitin-conjugating enzyme, E2. Whereas E2s can directly attach ubiquitin to lysine residues in a substrate, most physiological ubiquitination reactions probably require a ubiquitin ligase, or E3 (Hershko et al., J. Biol. Chem. 258:8206-8214, 1983). E3s have been implicated in substrate recognition and, in one case, transfer of ubiquitin from E2 to a substrate via an E3xcx9cubiquitin-thiolester intermediate (Scheffner et al., Nature 373:81-83, 1995). Once the substrate is multiubiquitinated, it is then recognized and degraded by the 26S proteasome.
A novel ubiquitination pathway has recently been discovered in budding yeast. Components of this pathway include the Cdc53, Cdc4, and Skp1 gene products, which assemble into a ubiquitin ligase complex known as SCFCdc4 (for Skp1, Cullin, F-box protein Cdc4). In yeast, SCF collaborates with the E2 enzyme Cdc34 to catalyze ubiquitination of the CDK inhibitor Sic1. The specificity of SCF is thought to be governed by Skp1 and the F-box-containing subunit Cdc4, which together form a substrate receptor that tethers Sic1 to the complex. The assembly of this receptor is thought to be mediated by a direct interaction between yeast Skp1 and the F-box domain of Cdc4 (Feldman et al., Cell 91:221-230, 1997; Skowyra et al., Cell 91:209-219, 1997).
Whereas genetic analysis has revealed that Sic1 proteolysis requires Cdc4, G1 cyclin proteolysis depends upon a distinct F-box-containing protein known as GRR1 (Barral et al., Genes Dev. 9:399-409, 1995). Alternative SCF complexes (SCFGRR1) assembled with GRR1 instead of Cdc4 bind G1 cyclins but not Sic1, suggesting that there exist multiple SCF complexes in yeast whose substrate specificities are dictated by the identity of the F-box subunit.
Components of the SCF ubiquitination pathway have been highly conserved during evolution. Human homologues of the yeast Cdc34 and Skp1 have been reported (Plon et al., Proc. Natl. Acad. Sci. USA 90:10484-10488, 1993; Zhang et al., Cell 82:912-925, 1995), and F-box-containing proteins like Cdc4 and GRR1 have been identified in many eukaryotes (Bai et al., Cell 86:263-274, 1996). Many of these F-box proteins also contain either WD-40 repeats (like Cdc4) or leucine-rich repeats (like GRR1). A potential human counterpart of GRR1, SKP2, has been identified along with human Skp1 as a Cyclin A/CDK2-associated protein that is necessary for S-phase progression (Zhang et al., Cell 82:912-925, 1995). Homologues of Cdc53, which are known as Cullins, are also present in many eukaryotes, including humans and nematodes (Kipreos et al., Cell 85:1-20, 1996; Mathias et al., Mol. Cell. Biol. 16:6634-6643, 1996).
It is currently thought that transitions from one phase of the cell cycle to another are coupled to fluctuations in the activity of a family of cyclin-dependent protein kinases (CDKs). These kinases represent a special family of kinases that are activated by regulatory proteins known as cyclins. Cyclins bind to the catalytic kinase subunit and trigger a battery of post-translational modifications that culminate in the activation of the kinase. Eventually, the kinase activity is extinguished by proteolysis of the stimulatory cyclin subunit. In yeast, a crucial means of regulating cell cycle progression is by the targeted degradation of both activating and inhibitory subunits of the cyclin dependent kinase Cdc28. The G1 to S phase transition is driven by the destruction of an inhibitor (Sic1) that restrains the activity of a cyclin/CDK complex that triggers DNA replication. The ubiquitin conjugating enzyme Cdc34 has been implicated in the ubiquitination of the regulatory proteins and their ultimate destruction (Goebl et al., Science 241:1331-5, 1988).
The two other proteins in the SCF complex, Cdc4 and Cdc53, have been found to be required for the G1 to S phase transition. The absence of functional Cdc4, Cdc34 or Cdc53 from the cell gives rise to identical terminal morphologies suggesting that these proteins interact to perform a function. Numerous genetic interactions are seen between these genes and the encoded proteins are found physically associated in vivo. Thus, the G1 to S phase transition in the yeast cell cycle requires the activity of a complex containing Cdc4, Cdc34 and Cdc53. Identification of counterparts of Cdc4, Cdc34, and Cdc53 in other species such as humans will provide new insights into how disturbances in ubiquitination influence diseases associated with cell proliferation.
Several human cell cycle regulators are targeted for ubiquitination following their phosphorylation by CDKs, implicating them as potential substrates of SCF pathway(s) in human cells. Among them is the CDK inhibitor p27, the abundance of which may be regulated by Cdc34-dependent ubiquitination (Pagano et al., Science 269:682-685, 1995; Sheaff, R. J., et al., Genes Dev. 11:1464-1478, 1997). In addition, Cyclins E and D1 are degraded by a ubiquitin-dependent pathway following phosphorylation at a specific site (e.g., Won and Reed, EMBO J. 15:4182-4193, 1996). It has also been suggested that cyclin A is a target of an SCF pathway. Alternatively, SCF-bound Cyclin A/CDK2 may phosphorylate SCF subunits or potential substrates such as E2F-1/DP-1, thereby activating SCF-dependent ubiquitination (Dynlacht et al., Genes Dev. 8:1772-1786, 1994).
Ubiquitination is also thought to play a role in tumor formation, as the ubiquitin system is associated with cell cycle regulation (King, R. W., et al., Science, 274:1652-9, 1996). For example, the protein E6, encoded by the human papilloma virus, which causes cervical cancer, was found to bind to a human ubiquitin-protein ligase, thereby targeting the tumor suppressor p53 for ubiquitin-mediated degradation (Scheffner et al, Cell 63:1129-36, 1990; Huibregtse et al., Molecular and Cellular Biology 13:775-84, 1993). A need exists, therefore, for assay systems that can identify modulators of ubiquitin ligase activity, some of which may be useful as therapeutic treatments. The present invention addresses this need.
The present invention is based on the discovery of a simplified assay for identifying modulators of ubiquitin ligase activity. This assay allows detection of compounds that affect ubiquitination and thus, cell cycle regulation in cells. Thus, the invention provides a method and assay system for identifying modulators of ubiquitin ligase activity comprising contacting a test sample composed of a ubiquitin ligase, an E1 enzyme, ubiquitin, ATP, and a substrate with a test compound under suitable test conditions and measuring the ubiquitination of the substrate. An increase in ubiquitination, in comparison to a test sample lacking a test compound, indicates a stimulation of activity, whereas a reduction in ubiquitination indicates an inhibitor of activity.
Also disclosed herein are methods of identifying proteins having ubiquitin ligase activity, methods of identifying substrates for ubiquitination, methods for identifying an activity relationship between a particular ubiquitin ligase and a particular ubiquitin conjugating enzyme, and chimeric proteins comprising a ubiquitin conjugating enzyme and a ubiquitination substrate, which are useful in all of the disclosed methods.